We propose to study the mode of action of calcitonin through analysis of receptor binding and biological effects of the hormone utilizing as target tissue, a recently characterized test system developed from work with a cultured porcine kidney cell line, LLC-PK1. We plan to follow important leads already gained through extensive preliminary studies. Results of these investigations indicate that our approaches have considerable promise not only for analysis of several aspects of the mode of action of this peptide hormone, but also should be applicable to understanding general mechanisms of polypeptide hormone interaction with target cells. Preliminary studies have indicated that the system is suitable not only for analysis of hormone-dependent cyclic AMP production but also a variety of biologic responses, including a highly specific pattern of proteolytic metabolism of calcitonin, hormone-specific changes in cell replication rate, cell size and surface morphology, and a hormone-dependent stimulation of synthesis of a specific protein, a plasminogen activator. We shall analyze the proteolytic metabolism of calcitonin, as revealed by microsequence analysis of calcitonin fragments produced during incubation with intact cells. In addition, we wish to determine whether all of the observed changes in cell function are dependent upon an initial stimulation of cyclic AMP production or, alternatively, whether certain biologic responses might be dependent instead on other messengers, such as changes in the rate of trans-membrane calcium flux since inhibition of calcium entry into these cells prevents a rise in plasminogen activator. Increased calcium entry may well involve interactions with the cellular calcium-binding protein calmodulin. Effects of structural modification of calcitonin on metabolism and biologic potency will be systematically evaluated. We will also study the nature and significance of the desensitization of LLC-PK1 cells which we have demonstrated occurs with exposure to calcitonin. We have found that desensitization results in decreased availability of receptor binding sites, but also in increased phosphodiesterase and membrane cyclase activity; therefore other biologic responses in desensitized cells will be studied.